Human immunodeficiency virus (HIV-1) Vpr induced downregulation of NHE1 induces alteration in intracellular pH and loss of ERM complex in target cells

Abstract

Human immunodeficiency virus type 1 (HIV-1) Vpr is known to dysregulate host cellular functions through its interaction with cellular proteins. Using a protein array we assessed Vpr-mediated differential regulation of host cellular proteins expression. Results demonstrated that Vpr differentially regulated host factors that are involved in functions, such as cell proliferation, differentiation and apoptosis. One of the most highly downregulated proteins attained was the sodium hydrogen exchanger, isoform 1 (NHE1), which showed a significant (60%) decrease in HIV-1 Vpr(+) virus infected cells as compared to HIV-1 Vpr(-) virus infected control. NHE1 downregulation further led to acidification of cells and was directly correlated with loss of ezrin, radixin and moesin (ERM) protein complex and decreased AKT phosphorylation. Vpr-mediated NHE1 dyregulation is in part through GR pathway as GR antagonist, mifepristone reversed Vpr-induced NHE1 downregulation.

Figure 1

Pathway analysis depicting cellular proteins down regulated by HIV-1 Vpr: Role in apoptosis.

Figure 2. Confirmation of NHE1 downregulation by western blot

Total PBMCs and macrophages from healthy donors or the cell line, CEM x174, were infected with HIV-1 vpr(+) or HIV-1 vpr(−) virus. (A) Percentage of cells infected by HIV-1 vpr(+) and HIV-1 vpr(−) virus was determined by flow cytometry: PBMCs infected as above were fixed, permeabilized, and stained for intracellular p24 antigen expression using FITC conjugated anti-p24 antibody. Histogram represents the HIV-1 vpr(+) virus infected (green) and HIV-1 vpr(−) virus infected cells (red). Histogram (gray) represents the isotype control (IgG-FITC) in infected cells. (B) Immunoblot analysis of NHE1 in HIV-1 vpr(+) or HIV-1 vpr(−) virus infected cell: Postinfection cells were lysed and subjected to immunoblot with the indicated antibodies. Anti-Vpr and anti-tubulin are shown as controls for Vpr expression and protein loading, respectively. Results are representative of six independent experiments.

Figure 3. Real time RT-PCR analysis of NHE1 expression

Healthy donor PBMCs infected with replication competent or AT-2 treated HIV-1 vpr(+) or HIV-1 vpr(−) virus or treated with Vpr (rVpr) or rGag (control) protein. RNA extracted from infected and control groups was reverse transcribed to cDNA and real time PCR was carried out using primers and probe specific for NHE1 or RPLPO. Ratios were generated using the ΔΔCT method of relative quantitation and values were normalized to the endogenous control RPLPO within each sample. In each experiment, the results attained in HIV-1 vpr(−) virus infected cells or rGag treated samples were considered as a ratio of 1.0. P values were based on results obtained from multiple experiments (N=5).

Figure 4. Intracellular pH in cells infected with HIV-1 vpr(+) or HIV-1 vpr(−) virus

PBMCs infected with HIV-1 vpr(+) or HIV-1 vpr(−) virus were used for measuring the intracellular pH. Cells were loaded with 10μM SNARF-1 in PBS at room temperature for 30 minutes in the dark. SNARF-1 was excited at 488nm and fluorescent intensity assessed at 575 and 645nm. (A) SNARF-1 calibration curve was generated using normal cells (B) Summary of the mean change in fluorescence intensity ratio and intracellular pH in HIV-1 vpr(+) versus HIV-1 vpr(−) virus infected cells (N=4).

Figure 5. (A) Effect of Vpr on ERM expression in infected cells

Confocal microscopy images of EGFP-reporter virus infected HeLa-T4 cells expressing ERM. Infected cells were directly visualized by EGFP (Green) and ERM by indirect immunofluorescence (Red). Arrows indicate virus-infected cells. The images are representative of three independent experiments. (B) Phospho-Akt in HIV-1 vpr(+) and HIV-1 vpr(−) virus infected PBMCs. Cell lysates generated from HIV-1 vpr(+) or HIV-1 vpr(−) virus infected cells were used in immunoblot for phospho-Akt and total Akt. Equivalent amount of protein (20μg) was separated by SDS-PAGE, transferred, and subjected to immunoblot with anti-phospho-Akt (ser 473) or anti-Akt. Alpha tubulin was used as a control for equivalent protein loading. Results are representative of four independent experiments.

Figure 5. (A) Effect of Vpr on ERM expression in infected cells

Confocal microscopy images of EGFP-reporter virus infected HeLa-T4 cells expressing ERM. Infected cells were directly visualized by EGFP (Green) and ERM by indirect immunofluorescence (Red). Arrows indicate virus-infected cells. The images are representative of three independent experiments. (B) Phospho-Akt in HIV-1 vpr(+) and HIV-1 vpr(−) virus infected PBMCs. Cell lysates generated from HIV-1 vpr(+) or HIV-1 vpr(−) virus infected cells were used in immunoblot for phospho-Akt and total Akt. Equivalent amount of protein (20μg) was separated by SDS-PAGE, transferred, and subjected to immunoblot with anti-phospho-Akt (ser 473) or anti-Akt. Alpha tubulin was used as a control for equivalent protein loading. Results are representative of four independent experiments.

Figure 6. Vpr mediated NHE1 downregulation is reversed by glucocorticoid antagonist, mifepristone

PBMCs were infected with HIV-1 vpr(+) or HIV-1 vpr(−) virus as described, and maintained in the presence or absence of different concentrations of mifepristone. Seventy-two hours post treatment cells were assessed for NHE1 mRNA expression via real-time RT-PCR. Figure represents the mean of three independent experiments and error bars represent standard deviation between experiments.